U.S. patent application number 17/547845 was filed with the patent office on 2022-06-23 for diagnosing heart disease and degenerative mitral valve disease in a canine.
The applicant listed for this patent is Societe des Produits Nestle S.A.. Invention is credited to Qinghong Li.
Application Number | 20220195483 17/547845 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-23 |
United States Patent
Application |
20220195483 |
Kind Code |
A1 |
Li; Qinghong |
June 23, 2022 |
DIAGNOSING HEART DISEASE AND DEGENERATIVE MITRAL VALVE DISEASE IN A
CANINE
Abstract
The present invention relates to methods for diagnosing heart
disease in a canine, including early stage degenerative mitral
valve disease, by using microbiome including specific genera and
species. In one embodiment, the method can comprise measuring a
normalized relative abundance of fecal bacteria including
Faecalibacterium, Turicibacter, Streptococcus, E. Coli, Blautia,
Fusobacterium, and C. hiranonis, calculating a dysbiosis index
based on the fecal bacteria, and determining that the canine has
heart disease if the dysbiosis index is greater than -1.0.
Inventors: |
Li; Qinghong; (Chesterfield,
MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Societe des Produits Nestle S.A. |
Vevey |
|
CH |
|
|
Appl. No.: |
17/547845 |
Filed: |
December 10, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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63127247 |
Dec 18, 2020 |
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International
Class: |
C12Q 1/06 20060101
C12Q001/06; C12Q 1/14 20060101 C12Q001/14 |
Claims
1. A method of diagnosing heart disease in a canine, comprising:
measuring a normalized relative abundance of fecal bacteria
including Faecalibacterium, Turicibacter, Streptococcus, E. Coli,
Blautia, Fusobacterium, and C. hiranonis; calculating a dysbiosis
index based on the fecal bacteria; and determining that the canine
has heart disease if the dysbiosis index is greater than -1.0.
2. The method of claim 1, further determining that the canine has
early stage degenerative mitral valve disease when the dysbiosis
index is between -1 and 0.5.
3. The method of claim 2, further determining that the early stage
degenerative mitral valve disease is B1 when the dysbiosis index is
between -1 and -0.25.
4. The method of claim 2, further determining that the early stage
degenerative mitral valve disease is B2 when the dysbiosis index is
between -0.25 and 0.5.
5. The method of claim 1, further determining that the canine has
congestive heart failure when the dysbiosis index is greater than
0.5.
6. A method of diagnosing early stage degenerative mitral valve
disease in a canine, comprising: measuring a normalized relative
abundance of a biomarker selected from the group consisting of
Catenibacterium mitsuokai, Butyricicoccus pulhcaecorum, Bacteroides
coprocola, Bacteroides plebeius, Allobaculum stercoricanis, or
combinations thereof; and determining that the canine has early
stage degenerative mitral valve disease if the normalized relative
abundance of Catenibacterium mitsuokai is between 0.3 to 3, the
normalized relative abundance of Butyricicoccus pulhcaecorum is
between 0.14 to 0.35, the normalized relative abundance of
Bacteroides coprocola is between 0.6 to 1.3, the normalized
relative abundance of Bacteroides plebeius is between 0.1 to 0.8,
or the normalized relative abundance of Allobaculum stercoricanisi
is between 0.1 and 1.5.
7. The method of claim 6, wherein the determining is based on at
least two biomarkers.
8. The method of claim 6, wherein the determining is based on at
least three biomarkers.
9. The method of claim 6, wherein the determining is based on at
least four biomarkers.
10. A method of diagnosing early stage degenerative mitral valve
disease in a canine, comprising: measuring a normalized relative
abundance of bacteria in a genus, wherein the genus is selected
from the group consisting of Catenibacterium, Prevotella,
Butyricicoccus, Faecalibacterium, Clostridium, Allobaculum, or
combinations thereof; and determining that the canine has early
stage degenerative mitral valve disease if the normalized relative
abundance of the bacteria in the Catenibacterium genus is between
0.3 to 3, the normalized relative abundance of the bacteria in the
Prevotella genus is between 0.5 to 4, the normalized relative
abundance of the bacteria in the Butyricicoccus genus is between
0.14 to 0.4, the normalized relative abundance of the bacteria in
the Faecalibacterium genus is between 0.012 to 0.04, the normalized
relative abundance of the bacteria in the Clostridium genus is
between 2 and 4, or the normalized relative abundance of the
bacteria in the Allobaculum genus is between 0.2 and 1.5.
11. The method of claim 10, wherein the determining is based on at
least two genera.
12. The method of claim 10, wherein the determining is based on at
least three genera.
13. The method of claim 10, wherein the determining is based on at
least four genera.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 63/127,247 filed Dec. 18, 2020, the disclosure
of which is incorporated in its entirety herein by this
reference.
BACKGROUND
[0002] Canine degenerative mitral valve disease (DMVD) is
characterized by slowly progressive valvular degeneration that
causes mitral regurgitation and, in some dogs, congestive heart
failure (CHF). Although dogs in the early stage typically have a
lengthy preclinical period, once progressed to the stage with CHF,
the disease advances more rapidly with a mean survival time less
than 12 months. Thus, it is of great interest to intervene at the
early preclinical stage to extend the longevity of affected dogs.
In recent years, a staging scheme for classifying canine DMVD has
been adopted by the consensus committee established by the American
College of Veterinary Internal Medicine (ACVIM). Dogs at risk of
developing DMVD but otherwise healthy are considered stage A; dogs
with a heart murmur due to mitral regurgitation but without
clinical signs of CHF are classified as stage B; dogs with overt
clinical signs of CHF are classified as stage C. Stage B dogs are
further divided into stage B1 or B2 due to the absence or presence
of cardiac remodeling.
[0003] Currently, the only medication that has been proven to be
effective for early preclinical DMVD is pimobanden, which, like any
pharmaceutical drug, comes with side effects. Currently, the gold
standard for DMVD diagnosis is echocardiogram, which is not only
expensive but also requires highly specialized veterinary
cardiologist. As such, effective diagnostic methods and treatments
overcoming the disadvantages of current methods and treatments
continue to be sought.
SUMMARY
[0004] The present disclosure relates generally to diagnosing heart
disease and degenerative mitral valve disease (DMVD) in a canine,
and in one aspect, early stage DMVD. In one embodiment, a method of
diagnosing heart disease in a canine can comprise measuring a
normalized relative abundance of fecal bacteria including
Faecalibacterium, Turicibacter, Streptococcus, E. Coli, Blautia,
Fusobacterium, and C. hiranonis, calculating a dysbiosis index
based on the fecal bacteria, and determining that the canine has
heart disease if the dysbiosis index is greater than -1.0.
[0005] In another embodiment, a method of diagnosing early stage
DMVD in a canine can comprise measuring a normalized relative
abundance of a biomarker selected from the group consisting of
Catenibacterium mitsuokai, Butyricicoccus pullicaecorum,
Bacteroides coprocola, Bacteroides plebeius, Allobaculum
stercoricanis, or combinations thereof; and determining that the
canine has early stage DMVD if the normalized relative abundance of
Catenibacterium mitsuokai is between 0.3 to 3, the normalized
relative abundance of Butyricicoccus pullicaecorum is between 0.14
to 0.35, the normalized relative abundance of Bacteroides coprocola
is between 0.6 to 1.3, the normalized relative abundance of
Allobaculum stercoricanisi is between 0.1 and 1.5.
[0006] In yet another embodiment, a method of diagnosing early
stage DMVD in a canine can comprise measuring a normalized relative
abundance of bacteria in a genus, wherein the genus is selected
from the group consisting of Catenibacterium, Prevotella,
Butyricicoccus, Faecalibacterium, Clostridium, Allobaculum, or
combinations thereof; and determining that the canine has early
stage DMVD if the normalized relative abundance of the bacteria in
the Catenibacterium genus is between 0.3 to 3, the normalized
relative abundance of the bacteria in the Prevotella genus is
between 0.5 to 4, the normalized relative abundance of the bacteria
in the Butyricicoccus genus is between 0.14 to 0.4, the normalized
relative abundance of the bacteria in the Faecalibacterium genus is
between 0.012 to 0.04, the normalized relative abundance of the
bacteria in the Clostridium genus is between 2 and 4, or the
normalized relative abundance of the bacteria in the Allobaculum
genus is between 0.2 and 1.5.
[0007] Additional features and advantages are described herein and
will be apparent from the following Detailed Description
DETAILED DESCRIPTION
[0008] Definitions
[0009] As used in this disclosure and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a fecal bacteria or bacterium" or "the fecal bacteria
or bacterium" includes two or more such bacteria or bacterium. The
term "and/or" used in the context of "X and/or Y" should be
interpreted as "X," or "Y," or "X and Y." Where used herein, the
terms "example" and "such as," particularly when followed by a
listing of terms, are merely exemplary and illustrative, and are
not exclusive or comprehensive.
[0010] As used herein, "about" is understood to refer to numbers in
a range of numerals, for example the range of -10% to +10% of the
referenced number, within -5% to +5% of the referenced number, or
in one aspect, within -1% to +1% of the referenced number, and in a
specific aspect, within -0.1% to +0.1% of the referenced number.
Furthermore, all numerical ranges herein should be understood to
include all integers, whole or fractions, within the range.
Moreover, these numerical ranges should be construed as providing
support for a claim directed to any number or subset of numbers in
that range. For example, a disclosure of from 1 to 10 should be
construed as supporting a range of from 1 to 8, from 3 to 7, from 1
to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.
[0011] As used herein, "between" is inclusive of the endpoints. For
example, a dysbiosis index between -1 and 0.5 includes where the
dysbiosis is -1 or 0.5.
[0012] All percentages expressed herein are by weight of the
composition on a dry matter basis unless specifically stated
otherwise. The skilled artisan will appreciate that the term "dry
matter basis" means that an ingredient's concentration or
percentage in a composition is measured or determined after any
free moisture in the composition has been removed. When reference
is made to the pH, values correspond to pH measured at 25.degree.
C. with standard equipment. An "amount" can be the total amount of
the referenced component per serving of the composition or per
distinct unit of the composition and/or can be the weight
percentage of the referenced component by dry weight. Moreover, an
"amount" includes zero; for example, the recitation of an amount of
a compound does not necessarily mean that the compound is present,
unless followed by a range that excludes zero.
[0013] As used herein, "normalized relative abundance" refers to
the amount of each microorganism calculated by taking each count
and dividing by the total sequence count in each sample and
transforming by square root.
[0014] As used herein, "early stage degenerative mitral valve
disease" refers to stage B of degenerative mitral valve
disease.
[0015] As used herein, "stage A" refers to dogs that are at risk of
developing degenerative mitral valve disease, but otherwise have a
healthy heart.
[0016] As used herein, "stage B" refers to dogs with a heart murmur
due to mitral regurgitation but without clinical signs of
congestive heart failure. "Stage B" includes stage B1 (absence of
cardiac remodeling and stage B2 (presence of cardiac
remodeling).
[0017] As used herein, stage C'' refer to dogs having congestive
heart failure.
[0018] As used herein, "degenerative mitral valve disease," "DMVD,"
"chronic valvular disease," "CVD," "myxomatous mitral valve
disease," and "MMVD" can be used interchangeably and refers to
progressive valvular degeneration that causes mitral regurgitation
and/or congestive heart failure (CHF) and includes stage A, stage
B, and stage
[0019] C.
[0020] As used herein, "dysbiosis index" or "DI" is quantified by
as a single numerical value that measured the closeness
(l.sub.2-norm) of the test sample to the mean (prototype) of each
class as disclosed in "A Dysbiosis Index to Assess Microbial
Changes in Fecal Samples of Dogs with Chronic Inflammatory
Enteropathy" by AlShawaqfeh et al, FEMS Microbiology Ecology, vol.
93, no. 11, pp 1-8 (2017) (doi:0.1093/femsec/fix136). As discussed
in the above article, DI is defined as the difference between
(Euclidean distance between the test sample and the healthy class
centroid) and the (Euclidean distance between the test and the
diseased class centroid). DI is calculated mathematically as
follows: the DI of a text sample z is defined as:
DI(z;.mu..sub.C.sub.D,
.mu..sub.C.sub.H)=.parallel.z-.mu..sub.C.sub.H.parallel..sub.2-.parallel.-
z-.mu..sub.C.sub.D.parallel..sub.2
[0021] Where .mu..sub.CD and .mu..sub.C.sub.H stand for the
centroid of the diseased and healthy samples in the training set,
respectively.
[0022] The methods disclosed herein may lack any step that is not
specifically disclosed herein. Thus, a disclosure of an embodiment
using the term "comprising" includes a disclosure of embodiments
"consisting essentially of" and "consisting of" the steps
identified. Any embodiment disclosed herein can be combined with
any other embodiment disclosed herein unless explicitly and
directly stated otherwise.
[0023] Unless defined otherwise, all technical and scientific terms
and any acronyms used herein have the same meanings as commonly
understood by one of ordinary skill in the art in the field of the
invention. Although any compositions, methods, articles of
manufacture, or other means or materials similar or equivalent to
those described herein can be used in the practice of the present
invention, the preferred compositions, methods, articles of
manufacture, or other means or materials are described herein.
[0024] All patents, patent applications, publications, and other
references cited or referred to herein are incorporated herein by
reference to the extent allowed by law. The discussion of those
references is intended merely to summarize the assertions made
therein. No admission is made that any such patents, patent
applications, publications or references, or any portion thereof,
are relevant prior art for the present invention and the right to
challenge the accuracy and pertinence of such patents, patent
applications, publications, and other references is specifically
reserved.
[0025] Embodiments
[0026] The present inventor has discovered that heart disease can
be diagnosed based on specific microbiome including a dysbiosis
index as well as specific genera and species. Further, the inventor
has discovered that the present microbiome can be used to diagnose
early stage degenerative mitral valve disease. Such methods allow
for an inexpensive and efficient diagnosis for conditions that can
be difficult to diagnose as well as costly.
[0027] In one embodiment, a method of diagnosing heart disease in a
canine can comprise measuring a normalized relative abundance of
fecal bacteria including Faecalibacterium, Turicibacter,
Streptococcus, E. Coli, Blautia, Fusobacterium, and C. hiranonis,
calculating a dysbiosis index based on the fecal bacteria, and
determining that the canine has heart disease if the dysbiosis
index is greater than -1.0.
[0028] Generally, the canine can be diagnosed with DMVD if the
dysbiosis index is greater than -1.0. However, in one aspect, the
determining step can also include determining that the canine has
early stage DMVD when the dysbiosis index is between -1 and 0.5.
Additionally, in another aspect, the present methods can further
determine that the canine has early stage DMVD B1 when the
dysbiosis index is between -1 and -0.25. In still another aspect,
the present methods can further determine that the canine has early
stage DMVD B2 when the dysbiosis index is between -0.25 and 0.5. In
yet another aspect, the present methods can further determine that
the canine has congestive heart failure when the dysbiosis index is
greater than 0.5.
[0029] In another embodiment, a method of diagnosing early stage
DMVD in a canine can comprise measuring a normalized relative
abundance of a biomarker selected from the group consisting of
Catenibacterium mitsuokai, Butyricicoccus pullicaecorum,
Bacteroides coprocola, Bacteroides plebeius, Allobaculum
stercoricanis, or combinations thereof; and determining that the
canine has early stage DMVD if the normalized relative abundance of
Catenibacterium mitsuokai is between 0.3 to 3, the normalized
relative abundance of Butyricicoccus pullicaecorum is between 0.14
to 0.35, the normalized relative abundance of Bacteroides coprocola
is between 0.6 to 1.3, the normalized relative abundance of
Bacteroides plebeius is between 0.1 to 0.8, or the normalized
relative abundance of Allobaculum stercoricanisi is between 0.1 and
1.5.
[0030] Generally, the canine can be diagnosed with early stage DMVD
using various biomarkers, i.e., bacteria, discussed herein having
specific normalized relative abundances. However, in one aspect,
the diagnosis can be based at least two biomarkers. In another
aspect, the diagnosis can be based on at least three biomarkers. In
still another aspect, the diagnosis can be based on at least four
biomarkers. In yet another aspect, the diagnosis can be based on
all five biomarkers.
[0031] In yet another embodiment, a method of diagnosing early
stage DMVD in a canine can comprise measuring a normalized relative
abundance of bacteria in a genus, wherein the genus is selected
from the group consisting of Catenibacterium, Prevotella,
Butyricicoccus, Faecalibacterium, Clostridium, Allobaculum, or
combinations thereof; and determining that the canine has early
stage DMVD if the normalized relative abundance of the bacteria in
the Catenibacterium genus is between 0.3 to 3, the normalized
relative abundance of the bacteria in the Prevotella genus is
between 0.5 to 4, the normalized relative abundance of the bacteria
in the Butyricicoccus genus is between 0.14 to 0.4, the normalized
relative abundance of the bacteria in the Faecalibacterium genus is
between 0.012 to 0.04, the normalized relative abundance of the
bacteria in the Clostridium genus is between 2 and 4, or the
normalized relative abundance of the bacteria in the Allobaculum
genus is between 0.2 and 1.5.
[0032] Generally, the canine can be diagnosed with early stage DMVD
using bacteria in various genera, discussed herein having specific
normalized relative abundances. However, in one aspect, the
diagnosis can be based at least two genera. In another aspect, the
diagnosis can be based on at least three genera. In still another
aspect, the diagnosis can be based on at least four genera. In
another aspect, the diagnosis can be based on at least five genera.
In yet another aspect, the diagnosis can be based on all six
biomarkers.
EXAMPLES
[0033] The following non-limiting examples are illustrative of
embodiments of the present disclosure.
[0034] Example 1-DMVD Study of Canines
[0035] Clinically healthy dogs 7 years of age or older without a
heart murmur and without concurrent systemic disease were
prospectively enrolled as controls (group A, N=29). A cohort of
dogs 7 years of age or older with a left apical systolic murmur,
echocardiographic (echo) diagnosis of thickened and prolapsing
mitral valve leaflet(s) and mitral regurgitation, as well as
clinical history and physical exam consistent with stage B1, B2, C,
or D DMVD were considered for group B1 (N=34), group B2 (N=25), and
group C/D (N=25), respectively. Any dog with severe concurrent
systemic disease including diabetes, cancer, or renal failure, or
those with any congenital heart disease were excluded. Dogs with
signs of gastrointestinal illness such as vomiting or diarrhea and
those that had received antibiotics within 30 days were also
excluded. Fecal samples were obtained from these dogs.
[0036] Diet intervention study with preclinical DMVD dogs
[0037] Dogs with preclinical DMVD and with body weight less than 15
kilograms were considered for inclusion in the study. Dogs were
randomly assigned to two diet groups, control diet (CON, N=9) and
diet supplemented with cardiac protect blend (CPB, N=10). The two
diets were previously described in details. Dog were fed their
assigned diets as their sole source of nutrition for 6 months.
Clinical measures and fecal samples were taken at baseline, 3 and 6
months.
[0038] Fecal DNA extraction and metagenomic sequencing
[0039] Fecal genomic DNA (Input-450-600 ng) was fragmented on the
Covaris LE220 instrument targeting 375 bp inserts. Automated
Illumina libraries were constructed with the KAPA Hyper PCR-free
library prep kit (KAPA Biosystems/Roche) on the SciClone NGS
platform (Perkin Elmer). The fragmented genomic DNA was size
selected on the SciClone instrument with AMPure XP beads to tighten
the distribution of DNA fragments to ensure the average insert of
the libraries were between 350-375 bps. The manufacturer's protocol
as provided by Perkin Elmer was followed, with the following
exception: Post ligation, the libraries were purified twice with a
0.7.times. AMPure bead/sample ratio to eliminate any residual
adaptors. An aliquot of the final libraries were diluted 1:5 and
quantitated on the Caliper GX instrument (Perkin Elmer). The
concentration of each library was accurately determined through
qPCR utilizing the KAPA library Quantification Kit according to the
manufacturer's protocol (KAPA Biosystems/Roche) to produce cluster
counts appropriate for Illumina NovaSeq6000 instrument. Libraries
were pooled and run over 0.1 of a NovaSeq6000 S4 flow cell using
the XP workflow and running a 150.times.10.times.10.times.150
sequencing recipe in accordance with manufacturer's protocol.
Approximately 5Gb paired-end sequences were generated for each
sample.
[0040] Dysbiosis index
[0041] The abundances of eight bacterial groups, including total
bacteria, Faecalibacterium, Turicibacter, Escherichia coli,
Streptococcus, Blautia, Fusobacterium and Clostridium hiranonis,
were evaluated on the fecal DNA samples from the four groups of
DMVD dogs: groups A (N=31), B1 (N=35), B2 (N=25), and C/D (N=30).
The qPCR primer sets, protocol and the method for dysbiosis index
(DI) were described previously in
[0042] AlShawaqfeh et al. FEMS Microbiology Ecology, vol. 93, no.
11, pp 1-8 (2017). A negative DI indicates normobiosis, whereas a
positive DI indicates dysbiosis. The reference interval for C.
hiranonis, a beneficial bacterium, was between 5.1-7.1.
[0043] Bioinformatics analysis
[0044] Quality of the sequences were examined using fastQC.
Paired-end sequences were stitched together using PEAR with default
settings. Trimmomatic was used to remove low quality sequences
while Bowtie2 was run to screen out contaminant sequences mapped to
the canine (CanFam3.1) or PhiX reference genomes. Low quality
sequences were scanned with a 4-base wide sliding window and
removed if the average quality score per base dropped below 20.
Sequences with less than 50 bases were also removed. Phylogenetic
analysis was performed using MetaPhlAn 2.0 and the abundance of
UniRef90 gene families and MetaCyc pathways was calculated using
HUMAnN 2.0.
[0045] Statistical analysis
[0046] For the cross-sectional study, multi-group comparisons were
performed using Kruskal-Wallis tests. Dunn's multiple comparisons
were performed on the significant taxa. For the diet intervention
study, changes from 3 months and 6 months over baseline were
calculated for each taxon. Then the differences between group means
(CPB vs. CON) were calculated. A positive number indicates an
increase in CPB from CON, while a negative number indicates a
decrease.
[0047] Results
[0048] Table 1 provides bacterial species with differential
abundances among groups A, B1, B2, and C/D, which refer to the four
stages of canine DMVD. Table 2 provides changes between CPB and CON
bacterial abundance at 3 months and 6 months, which were normalized
over baseline values with means calculated. Twenty-three bacterial
species were changes among the four groups of DMVD dogs (Table 1).
Among them, seven bacteria, B. plebeiu, A. stercoricanis, E.
biforme, B. coprocola, B. pullicaecorum, C. mitsuokai, and P.
copri, whose abundances were decreased with DMVD severity had
increased abundances in CPB-fed dogs vs. CON-fed dogs, while the
abundance of Bacteroides vulgatus was reduced in CPB-fed dogs vs.
CON-fed dogs (Table 2).
TABLE-US-00001 TABLE 1 P Mean Genus Species value A B1 B2 C/D
Lactococcus lactis 0.0008 0 0.001 0.003 0.054 Streptococcus minor
0.0019 0.002 0.002 0.002 0.033 Subdoligranulum unclassified 0.0018
0.014 0.008 0 0.049 Catenibacterium mitsuokai 0.0011 3.994 1.494
0.319 0.233 Eubacterium biforme 0.0022 1.299 0.383 0.047 0.071
Prevotella copri 0.0046 5.265 1.478 1.628 0.261 Butyricicoccus
pullicaecorum 0.0042 0.455 0.256 0.152 0.134 Flavonifractor plautii
0.0075 0.008 0.001 0 0.02 Bacteroides coprocola 0.0096 1.925 0.918
0.811 0.44 Bacteroides plebeius 0.0137 0.934 0.727 0.277 0.057
Lactococcus garvieae 0.0126 0 0 0.002 0 Faecalibacterium
prausnitzii 0.0148 0.048 0.013 0.015 0.01 Escherichia coli 0.0165
1.381 2.031 5.269 2.875 Escherichia unclassified 0.0224 0.706 0.585
2.274 1.358 Bacteroides vulgatus 0.0273 1.077 1.419 3.774 0.982
Parabacteroides distasonis 0.0301 0 0.034 0.021 0.373 Turicibacter
unclassified 0.0299 0.514 0 0 0 Clostridium perfringens 0.0422
0.087 0.701 2.659 2.054 Allobaculum stercoricanis 0.0429 1.93 1.095
0.807 0.063 Bilophila unclassified 0.0404 0 0 0.143 0.095 Proteus
mirabilis 0.0435 0 0.008 0 0.163 Streptococcus infantarius 0.048
0.001 0.026 0 0 Lachnospiraceae bacterium 7 1 0.048 0 0 0 0.002
58FAA
TABLE-US-00002 TABLE 2 3 months - baseline CPB - 6 months -
baseline CPB - Genus Species CON CPB CON CON CPB CON Bacteroides
plebeius 0.0813 0.6817 0.6 0.0813 0.6817 0.6 Allobaculum
stercoricanis -0.9075 -0.1596 0.7 -2.4208 -1.123 1.3 Eubacterium
biforme -1.3652 -0.4872 0.9 -1.4233 -0.6205 0.8 Bacteroides
coprocola 0.0018 0.712 0.7 0.5066 2.7123 2.2 Butyricicoccus
pullicaecorum 0.0616 0.0184 0 -0.1575 -0.0084 0.1 Catenibacterium
mitsuokai -3.5703 -0.2561 3.3 -3.6665 -1.179 2.5 Prevotella copri
0.7739 8.8631 8.1 12.5194 17.827 5.3 Bacteroides vulgatus 1.6525
0.2789 -1.4 0.5871 0.3112 -0.3
[0049] Table 3 provides bacterial genus with differential
abundances among groups A, B1, B2, and C/D, which refer to the four
stages of canine DMVD. Table 3 provides changes between CPB and CON
bacterial abundance at 3 months and 6 months, which were normalized
over baseline values with means calculated. Three bacterial genera,
Prevotella, Catenibacterium, and Allobaculum, had decreased
abundances with DMVD severity (Table 3), but their abundances were
increased in dogs fed CPB diet vs. CON diet (Table 4).
TABLE-US-00003 TABLE 3 Genus P value A B1 B2 C/D Subdoligranulum
0.0018 0.014 0.008 0 0.049 Catenibacterium 0.0011 3.994 1.494 0.319
0.233 Prevotella 0.0046 5.265 1.478 1.628 0.261 Lactococcus 0.0031
0 0.001 0.005 0.054 Butyricicoccus 0.0042 0.455 0.256 0.152 0.134
Flavonifractor 0.0075 0.008 0.001 0 0.02 Escherichia 0.0116 2.086
2.616 7.542 4.233 Faecalibacterium 0.0148 0.048 0.013 0.015 0.01
Clostridium 0.0388 1.768 2.692 3.832 4.834 Allobaculum 0.0429 1.93
1.095 0.807 0.063 Bilophila 0.0404 0 0 0.163 0.096 Proteus 0.0435 0
0.008 0 0.163
TABLE-US-00004 TABLE 4 3 months - baseline CPB - 6 months -
baseline CPB - Genus CON CPB CON CON CPB CON Prevotella 0.7739
8.8631 8.1 12.5194 17.827 5.3 Catenibacterium -3.5703 -0.2561 3.3
-3.6665 -1.179 2.5 Allobaculum -0.9075 -0.1596 0.7 -2.4208 -1.123
1.3
[0050] The DI (log DNA/gram of feces) for group A healthy dogs was
-1.48, but was increased to -0.6, -0.07 in groups B1 and B2
preclinical DMVD dogs respectively, and to 1.47 in group C/D dogs
with congestive heart failure. Thus, the DI can be used as an early
indicator of DMVD in dogs (Table 5). In addition, the abundance of
Clostridium hiranonis, a beneficial bacterial that converts primary
bile acids to secondary bile acids, was within the reference
interval in healthy dogs, but outside the interval in dogs with
DMVD (Table 5).
TABLE-US-00005 TABLE 5 Mean (log DNA/gram of feces) A B1 B2 C/D
Reference interval Dysbiosis Index -1.48 -0.6 -0.07 1.47 <0
normobiosis; >0 dysbiosis Total bacteria 11.28 11.33 11.31 11.24
10.6-11.4 Faecalibacterium 6.05 5.39 5.14 5.1 3.4-8.0 Turicibacter
7.18 6.74 6.92 6.4 4.6-8.1 Streptococcus 6.13 6.43 5.83 6.55
1.9-8.0 E. Coli 4.39 4.43 5.67 5.64 0.9-8.0 Blautia 11.03 11.35
11.23 11.32 9.5-11.0 Fusobacterium 8.7 8.61 8.88 8.18 7.0-10.3 C.
hiranonis 5.12 5.06 4.43 3.9 5.1-7.1
[0051] It should be understood that various changes and
modifications to the presently preferred embodiments described
herein will be apparent to those skilled in the art. Such changes
and modifications can be made without departing from the spirit and
scope of the present subject matter and without diminishing its
intended advantages. It is therefore intended that such changes and
modifications be covered by the appended claims.
* * * * *